chemosensors

Article

A Voltammetric Sensor Based on Chemically ReducedGraphene Oxide-Modified Screen-Printed CarbonElectrode for the Simultaneous Analysis of Uric Acid,Ascorbic Acid and Dopamine

Prosper Kanyong *, Sean Rawlinson and James Davis

School of Engineering, Ulster University, Jordanstown BT37 0QB, United Kingdom;Rawlinson-S@email.ulster.ac.uk (S.R.); james.davis@ulster.ac.uk (J.D.)* Correspondence: p.kanyong@ulster.ac.uk; Tel.: +44-28-9036-8663

Academic Editor: Igor MedintzReceived: 28 November 2016; Accepted: 17 December 2016; Published: 20 December 2016

Abstract: A disposable screen-printed carbon electrode (SPCE) modified with chemically reducedgraphene oxide (rGO) (rGO-SPCE) is described. The rGO-SPCE was characterized by UV-Visand electrochemical impedance spectroscopy, and cyclic voltammetry. The electrode displaysexcellent electrocatalytic activity towards uric acid (UA), ascorbic acid (AA) and dopamine (DA).Three resolved voltammetric peaks (at 183 mV for UA, 273 mV for AA and 317 mV for DA,all vs. Ag/AgCl) were found. Differential pulse voltammetry was used to simultaneously detect UA,AA and DA in their ternary mixtures. The linear working range extends from 10 to 3000 µM for UA;0.1 to 2.5 µM, and 5.0 to 2 × 104 µM for AA; and 0.2 to 80.0 µM and 120.0 to 500 µM for DA, and thelimits of detection (S/N = 3) are 0.1, 50.0, and 0.4 µM, respectively. The performance of the sensorwas evaluated by analysing spiked human urine samples, and the recoveries were found to be wellover 98.0% for the three compounds. These results indicate that the rGO-SPCE represents a sensitiveanalytical sensing tool for simultaneous analysis of UA, AA and DA.

Keywords: multiplex sensing; graphene-based electrodes; differential pulse voltammetry;electrochemical impedance spectroscopy; UV-Vis spectroscopy; screen-printed electrodes

1. Introduction

Uric acid (UA) is a principal end product of purine metabolism [1] and abnormal levels in urineand/or blood are symptomatic of diseases such as Lesch–Nyhan syndrome, gout and hyperuricemia,and renal and cardiovascular diseases [2]. Ascorbic acid (AA) is a water-soluble vitamin that hasantioxidant properties and can be found in high concentrations in some plant foods [3]. AA isinvolved in many biological processes including improving immunity, cancer prevention, treatingcommon cold, scurvy and mental illness, and free radical scavenging [4]. Dopamine (DA) is anessential neurotransmitter that forms part of the family of catecholamines. It plays vital roles in thenormal functions of the renal, metabolic, hormonal, cardiovascular and central nervous systems [5,6].High levels of DA in human bodily fluids have been found to be associated with several neurologicaldisorders including Huntington’s disease, restless legs syndrome, schizophrenia, attention deficithyperactivity disorder and Parkinson’s disease [7,8].

Several analytical methods such as high performance liquid chromatography, chemiluminescence,UV-Vis spectroscopy, capillary electrophoresis and electroanalytical methods [9–11] have beendeveloped for the detection of these compounds. Owing to the electroactive nature of UA, AA andDA, and their coexistence in human bodily fluids such as urine, electrochemical method is consideredmost attractive for their simultaneous analysis because they are rapid, low-cost and sensitive.

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However, the main drawbacks associated with the use of traditional electrode materials for theirsimultaneous analysis are low reproducibility due to electrode fouling and poor selectivity arising fromoverlapping voltammetric peaks [12]. Electrodes modified with nanoparticles [13], ionic liquids [14],redox mediators [15], polymers and polymer composites [16], metal oxides [17], and carbon black,carbon nanotubes or fullerene [18] have been useful in solving these problems. However, there arestill some drawbacks with regards to electrode complexity and inactivation, as well as stability ofelectrode modifiers/particles; thus, it is important to find suitable electrode materials that permit theirselective determination.

In this study, reduced graphene oxide (rGO) was prepared through mild treatment of grapheneoxide (GO) with sodium borohydride (NaBH4). The rGO dispersion then was casted onto theworking area of an in-house fabricated screen-printed carbon electrode (SPCE) to form rGO-SPCE.The electrochemical behaviour of UA, AA and DA at rGO-SPCE was thoroughly investigated.The sensor was subsequently employed for the simultaneous analysis of the compounds in ternarymixtures in buffer. Details of the sensor fabrication, characterization and application to the analysis ofthe compounds are described and discussed.

2. Experimental

2.1. Apparatus and Reagents

Electrochemical experiments were conducted using VSP-300 MultichannelPotentiostat/Galvanostat/EIS (Bio-Logic Science Instruments, Seyssinet-Pariset, France) witha standard three-electrode configuration. Impedance measurements in K4[Fe(CN)6] was carriedout at open circuit from frequency range of 200 kHz to 0.1 Hz. Under the optimized conditions,unless otherwise stated, DPVs were recorded with pulse amplitude of 50 mV, pulse time of 0.05 s,voltage step time of 0.5 s, voltage step of 5 mV and at 50 mV·s−1 scan rate. UV-Vis spectra wererecorded using Jenway 6715 UV-Vis (Multi-cell changer) spectrophotometer (Bibby Scientific Ltd.,Stone, UK). An Ag/AgCl (1.0 M KCl), SPCE or rGO-SPCE and a platinum wire were used asthe reference, working and counter electrodes. Ascorbic acid and uric acid were purchased fromAlfa Aesar, Heysham, UK. Graphene oxide (GO), dopamine hydrochloride, methanol, NaOH,NaBH4 and sodium carbonate (Na2CO3) were purchased from Sigma Aldrich, Irvine, UK. Urinesamples were obtained from the authors and informed consent was obtained.

2.2. Preparation of rGO

The chemical reduction of GO was per the method reported by Chua and Pumera [19] with somemodification. Briefly, to a dispersion of graphene oxide (6.0 mg) in methanol (2.5 mL) was addedNa2CO3 (5.0 mM) to adjust the pH to ~8.5. Sodium borohydride (NaBH4, 28.4 mg) was then addedinto the resultant dispersion and stirred at 70 C with refluxing for 5 hr. Upon completion, the mixturewas repeatedly centrifuged (at 400 rpm, 15 min) and washed with methanol and water until a neutralpH of the filtrate was obtained. The filtrate (reduced graphene oxide, rGO) was then re-suspended in2.0 mL of distilled water.

2.3. Fabrication of SPCE and rGO-SPCE

The fabrication of the unmodified SPCE transducer was prepared as previously described [20,21].Briefly, the base unmodified SPCE transducer was printed onto a Valox substrate using DEK240 Manual Screen Printing Machine, Stainless Steel Screen Mesh and graphite ink (GEM Productcode: C205010697) and cured at 70 C for 90 min. A polymeric dielectric material (GEM Product code:D2071120P1) was then screen-printed onto the cured SPCE to define the working area. Thereafter,2.0 µL of rGO suspension in water was dropped onto the working area of the SPCE and allowed toair-dry in room temperature to form rGO-SPCE (Scheme 1). The rGO-SPCE was rinsed in distilledwater to remove any unbound rGO and dried in N2.

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Scheme 1. (A) Screen-printing of carbon electrodes; and (B) chemical reduction of graphene oxide

and modification of SPCE.

3. Results and Discussion

3.1. Characterization of rGO-SPCE

Both graphene oxide (GO) and reduced graphene oxide (rGO) possess unique set of electrical

and optical properties that is different from pristine graphene because of the structural changes

arising from the presence of oxygen functionalities within sp2 bonded carbon networks [17,22].

Analyses of GO and rGO were performed using UV-Vis spectroscopy (Figure 1A). The GO spectrum

exhibits a characteristic absorption peak at 235 nm corresponding to the π→π* transition of the

aromatic C=C bonds [22]. After chemical reduction with NaHB4, this peak shifts from 254 nm to

282 nm, and the intensity of absorption in the spectra region increased, indicating that the conjugated

C=C bonds were restored by reduction [23]; thus, giving evidence that the GO has been reduced.

Scheme 1. (A) Screen-printing of carbon electrodes; and (B) chemical reduction of graphene oxide andmodification of SPCE.

3. Results and Discussion

3.1. Characterization of rGO-SPCE

Both graphene oxide (GO) and reduced graphene oxide (rGO) possess unique set of electrical andoptical properties that is different from pristine graphene because of the structural changes arisingfrom the presence of oxygen functionalities within sp2 bonded carbon networks [17,22]. Analyses ofGO and rGO were performed using UV-Vis spectroscopy (Figure 1A). The GO spectrum exhibits acharacteristic absorption peak at 235 nm corresponding to the π→π* transition of the aromatic C=Cbonds [22]. After chemical reduction with NaHB4, this peak shifts from 254 nm to 282 nm, and theintensity of absorption in the spectra region increased, indicating that the conjugated C=C bonds wererestored by reduction [23]; thus, giving evidence that the GO has been reduced.

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Scheme 1. (A) Screen-printing of carbon electrodes; and (B) chemical reduction of graphene oxide

and modification of SPCE.

3. Results and Discussion

3.1. Characterization of rGO-SPCE

Both graphene oxide (GO) and reduced graphene oxide (rGO) possess unique set of electrical

and optical properties that is different from pristine graphene because of the structural changes

arising from the presence of oxygen functionalities within sp2 bonded carbon networks [17,22].

Analyses of GO and rGO were performed using UV-Vis spectroscopy (Figure 1A). The GO spectrum

exhibits a characteristic absorption peak at 235 nm corresponding to the π→π* transition of the

aromatic C=C bonds [22]. After chemical reduction with NaHB4, this peak shifts from 254 nm to

282 nm, and the intensity of absorption in the spectra region increased, indicating that the conjugated

C=C bonds were restored by reduction [23]; thus, giving evidence that the GO has been reduced.

Figure 1. Cont.

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Figure 1. (A) UV-Vis spectra of GO and rGO suspensions in water. (B) Nyquist plots observed for

impedance measurements at SPCE and rGO-SPCE in Britton–Robinson (BR) buffer (pH 7.0)

containing 5.0 mM K4[Fe(CN)6] and 0.1 M KCl. Insert: Randles equivalent circuit used for data fitting,

Rs, resistance of the electrolyte solution; RCT, electron-transfer resistance; Zw, Warburg impedance; Cdl,

double layer capacitance. (C) Cyclic voltammograms of 5.0 mM K4[Fe(CN)6] in BR buffer (pH 7.0)

containing 0.1 M KCl at SPCE and rGO-SPCE, 50.0 mV·s−1 scan rate.

The electrochemical properties of SPCE and rGO-SPCE were examined using electrochemical

impedance spectroscopy (EIS) and cyclic voltammetry. EIS is a versatile and sensitive technique for

studying charge transfer processes occurring at electrode surfaces [24]. Figure 1B illustrates

experimental Nyquist spectra recorded in K4[Fe(CN)6] solution using SPCE and rGO-SPCE including

the equivalent Randles circuit used to extract the impedance circuit fitted data in Table 1. According

to the Randles circuit, a semi-circle located at the higher frequency region, corresponds to the charge

transfer resistance (RCT) of the electrode material. The SPCE showed RCT value of ~16 kΩ while

rGO-SPCE had a reduced RCT value of ~5.0 kΩ. This shows that the charge transfer rate for

[Fe(CN)6]3−/[Fe(CN)6]4− redox process increased upon employing rGO. From the obtained data

(Table 1), it can be concluded that rGO-SPCE have higher electrocatalytic activity in comparison with

the bare SPCE.

Table 1. Impedance circuit fitted parameters for SPCE and rGO-SPCE.

Parameter SPCE rGO-SPCE

Rs (Ω) 2998 2997

Cdl (μF) 1.4986 7.071

RCT (Ω) 16049 5004

Zw (Ω.s−½ ) 2181 1983

Cyclic voltammetry (CV) was also carried out as an independent method to verify the findings

of the EIS. Figure 1C shows CVs of K4[Fe(CN)6] at SPCE and rGO-SPCE. In comparison to what

occurred at SPCE, rGO-SPCE did not only exhibit well-defined redox peaks but also exhibited a

characteristic increase of the anodic and cathodic peaks for [Fe(CN)6]3−/[Fe(CN)6]4− redox couple.

Higher peak currents and a smaller peak-to-peak potential separation (ΔEp) at rGO-SPCE (Ipa = 18.5 μA,

Ipc = 14.8 μA; ΔEp = ~140.0 mV) when compared with SPCE (Ipa = 2.9 μA, Ipc = 1.1 μA; ΔEp= ~500 mV)

were observed. There were also slight shifts in both the anodic and cathodic peak potentials to less

positive values, giving rise to a smaller peak-to-peak separation (ΔEp = 140.0 mV). This enhancement

in electrocatalytic activity can be attributed to the electrocatalytic effect of rGO. Thus, the analysed

CV data are in agreement with the results obtained from EIS and provides further evidence of the

reduction of GO.

3.2. Cyclic Voltammetric Analysis of UA, AA and DA at SPCE and rGO-SPCE

Typical CVs recorded for a ternary mixture of UA, AA and DA at SPCE and rGO-SPCE in

Britton–Robinson (BR) buffer (pH 7.0) are depicted in Figure 2. In Figure 2A, UA and AA exhibit

broad irreversible oxidation peaks at 0.135 V and 0.295 V on SPCE, respectively, while DA has an

Figure 1. (A) UV-Vis spectra of GO and rGO suspensions in water. (B) Nyquist plots observedfor impedance measurements at SPCE and rGO-SPCE in Britton–Robinson (BR) buffer (pH 7.0)containing 5.0 mM K4[Fe(CN)6] and 0.1 M KCl. Insert: Randles equivalent circuit used for data fitting,Rs, resistance of the electrolyte solution; RCT, electron-transfer resistance; Zw, Warburg impedance;Cdl, double layer capacitance. (C) Cyclic voltammograms of 5.0 mM K4[Fe(CN)6] in BR buffer (pH 7.0)containing 0.1 M KCl at SPCE and rGO-SPCE, 50.0 mV·s−1 scan rate.

The electrochemical properties of SPCE and rGO-SPCE were examined using electrochemicalimpedance spectroscopy (EIS) and cyclic voltammetry. EIS is a versatile and sensitive techniquefor studying charge transfer processes occurring at electrode surfaces [24]. Figure 1B illustratesexperimental Nyquist spectra recorded in K4[Fe(CN)6] solution using SPCE and rGO-SPCE includingthe equivalent Randles circuit used to extract the impedance circuit fitted data in Table 1. According tothe Randles circuit, a semi-circle located at the higher frequency region, corresponds to the chargetransfer resistance (RCT) of the electrode material. The SPCE showed RCT value of ~16 kΩ whilerGO-SPCE had a reduced RCT value of ~5.0 kΩ. This shows that the charge transfer rate for[Fe(CN)6]3−/[Fe(CN)6]4− redox process increased upon employing rGO. From the obtained data(Table 1), it can be concluded that rGO-SPCE have higher electrocatalytic activity in comparison withthe bare SPCE.

Table 1. Impedance circuit fitted parameters for SPCE and rGO-SPCE.

Parameter SPCE rGO-SPCE

Rs (Ω) 2998 2997Cdl (µF) 1.4986 7.071RCT (Ω) 16049 5004

Zw (Ω·s−1/2) 2181 1983

Cyclic voltammetry (CV) was also carried out as an independent method to verify the findingsof the EIS. Figure 1C shows CVs of K4[Fe(CN)6] at SPCE and rGO-SPCE. In comparison to whatoccurred at SPCE, rGO-SPCE did not only exhibit well-defined redox peaks but also exhibiteda characteristic increase of the anodic and cathodic peaks for [Fe(CN)6]3−/[Fe(CN)6]4− redoxcouple. Higher peak currents and a smaller peak-to-peak potential separation (∆Ep) at rGO-SPCE(Ipa = 18.5 µA, Ipc = 14.8 µA; ∆Ep = ~140.0 mV) when compared with SPCE (Ipa = 2.9 µA, Ipc = 1.1 µA;∆Ep= ~500 mV) were observed. There were also slight shifts in both the anodic and cathodic peakpotentials to less positive values, giving rise to a smaller peak-to-peak separation (∆Ep = 140.0 mV).This enhancement in electrocatalytic activity can be attributed to the electrocatalytic effect of rGO.Thus, the analysed CV data are in agreement with the results obtained from EIS and provides furtherevidence of the reduction of GO.

3.2. Cyclic Voltammetric Analysis of UA, AA and DA at SPCE and rGO-SPCE

Typical CVs recorded for a ternary mixture of UA, AA and DA at SPCE and rGO-SPCE inBritton–Robinson (BR) buffer (pH 7.0) are depicted in Figure 2. In Figure 2A, UA and AA exhibit broad

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irreversible oxidation peaks at 0.135 V and 0.295 V on SPCE, respectively, while DA has an anodicpeak and a broadened cathodic peak at 0.295 V and 0.050 V (∆Ep = ~245 mV), respectively. This is anindication of a sluggish electron transfer process, which can be attributed to the conductivity of theSPCE material and/or electrode fouling caused by the deposition of the compounds including theirredox products on the electrode. The oxidation peaks for AA and DA overlaps; thus, the oxidationpeaks associated with the compounds could not be resolved. However, at rGO-SPCE, the oxidationpeaks for UA, AA and DA are resolved at ~0.112 V, ~0.18 V and 0.287 V, respectively. The oxidation peakfor DA (at ~0.287 V) formed a quasi-reversible couple at ~0.101 V. This anodic peak can be attributed tothe oxidation of DA to o-dopaminequinone while the cathodic peak can be attributed to the reductionof the o-dopaminequinone back to DA as has been previously reported [25]. The smaller ∆Ep valueof 186 mV (when compared with 245 mV at SPCE) for DA suggests that the reversibility of DA atrGO-SPCE is remarkably improved; thus, enabling the simultaneous analysis of the three compounds.

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anodic peak and a broadened cathodic peak at 0.295 V and 0.050 V (ΔEp = ~245 mV), respectively.

This is an indication of a sluggish electron transfer process, which can be attributed to the

conductivity of the SPCE material and/or electrode fouling caused by the deposition of the

compounds including their redox products on the electrode. The oxidation peaks for AA and DA

overlaps; thus, the oxidation peaks associated with the compounds could not be resolved. However,

at rGO-SPCE, the oxidation peaks for UA, AA and DA are resolved at ~0.112 V, ~0.18 V and 0.287 V,

respectively. The oxidation peak for DA (at ~0.287 V) formed a quasi-reversible couple at ~0.101 V.

This anodic peak can be attributed to the oxidation of DA to o-dopaminequinone while the cathodic

peak can be attributed to the reduction of the o-dopaminequinone back to DA as has been previously

reported [25]. The smaller ΔEp value of 186 mV (when compared with 245 mV at SPCE) for DA

suggests that the reversibility of DA at rGO-SPCE is remarkably improved; thus, enabling the

simultaneous analysis of the three compounds.

Figure 2. Cyclic voltammograms of ternary mixtures of UA (0.5 mM), AA (5.0 mM) and DA (0.5 mM)

in BR buffer (pH 7.0) at: (A) SPCE; and (B) rGO-SPCE. Scan rate, 50 mV·s−1.

3.3. The Effect of Scan Rate on Voltammetric Behavior of UA, AA and DA at rGO-SPCE

The kinetics of the electrode reaction was further investigated by evaluating the effect of scan

rate (v) on the anodic peak currents (Ipa). As shown in Figure 3, increasing scan rate increases the Ipa

of UA, AA and DA linearly with slight positive shifts in the oxidation peak potentials (Epa),

suggesting that adsorption of the compounds on rGO-SPCE does not occur. Plots of Ipa vs. square

root of scan rate (√v) (shown as inserts in Figure 3) resulted in the following linear equations: UA,

Ipa (μA) = 8.99 v1/2 (mV·s−1) − 30.89, R2 = 0.994; AA, Ipa (μA) = 10.59 v1/2 (mV·s−1) − 39.41, R2 = 0.977; and

DA, Ipa (μA) = 12.38 v1/2 (mV·s−1) − 40.88, R2 = 0.999. These suggest a diffusion-controlled process.

Figure 2. Cyclic voltammograms of ternary mixtures of UA (0.5 mM), AA (5.0 mM) and DA (0.5 mM)in BR buffer (pH 7.0) at: (A) SPCE; and (B) rGO-SPCE. Scan rate, 50 mV·s−1.

3.3. The Effect of Scan Rate on Voltammetric Behavior of UA, AA and DA at rGO-SPCE

The kinetics of the electrode reaction was further investigated by evaluating the effect of scan rate(v) on the anodic peak currents (Ipa). As shown in Figure 3, increasing scan rate increases the Ipa of UA,AA and DA linearly with slight positive shifts in the oxidation peak potentials (Epa), suggesting thatadsorption of the compounds on rGO-SPCE does not occur. Plots of Ipa vs. square root of scan rate(√

v) (shown as inserts in Figure 3) resulted in the following linear equations: UA, Ipa (µA) = 8.99 v1/2

(mV·s−1) − 30.89, R2 = 0.994; AA, Ipa (µA) = 10.59 v1/2 (mV·s−1) − 39.41, R2 = 0.977; and DA,Ipa (µA) = 12.38 v1/2 (mV·s−1) − 40.88, R2 = 0.999. These suggest a diffusion-controlled process.

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Figure 3. Cyclic voltammograms recorded at rGO-SPCE in BR buffer (pH 7.0) containing: (A) 0.5 mM

UA; (B) 5.0 mM AA; and (C) 0.5 mM DA, at scan rates of: (a) 10; (b) 50; (c) 100; (d) 150; and

(e) 200 mV·s−1. Inserts: Ipa vs. √v.

3.4. The Effect of pH on Voltammetric Behavior of UA, AA and DA at rGO-SPCE

The effect of pH on Ipa and Epa were also examined in the range of 3.0–10.0. As shown in

Figure 4A, when the pH of the solution was increased, Ipa increased gradually and then decayed with

the optimum pH being 7.0. Moreover, considering the fact that physiological pH is around 7.0 [26],

BR buffer solution of pH 7.0 was used as the supporting electrolyte for the analyses of the

compounds. Figure 4B illustrates the effect of pH on Epa, which followed the linear regression

equations E𝑝𝑎(𝑉) = 0.51 − 0.06 𝑝𝐻, R² = 0.9998 for UA, E𝑝𝑎(𝑉) = 0.45 − 0.04 𝑝𝐻, R² = 0.9996 for

AA, and E𝑝𝑎(𝑉) = 0.72 − 0.06 𝑝𝐻, R² = 0.9995 for DA. The Epa shifted with every increase of the pH

value of the supporting electrolyte for all the three compounds, suggesting that the reactions at

rGO-SPCE were accompanied by proton transfers [27]. The slope values of 60.0 mV·pH−1 for both UA

and DA suggest that their electrocatalytic reactions involved the transfer of equal number of protons

and electrons [28]. However, the slope value of 40.0 mV·pH−1 for AA is similar to the value given by

the Nernstian equation for reactions involving unequal electron and proton transfers [29]; thus, the

overall reaction of AA at rGO-SPCE could be classified as an electrochemical process that is followed

by a chemical reaction [30].

Figure 3. Cyclic voltammograms recorded at rGO-SPCE in BR buffer (pH 7.0) containing: (A) 0.5 mMUA; (B) 5.0 mM AA; and (C) 0.5 mM DA, at scan rates of: (a) 10; (b) 50; (c) 100; (d) 150;and (e) 200 mV·s−1. Inserts: Ipa vs.

√v.

3.4. The Effect of pH on Voltammetric Behavior of UA, AA and DA at rGO-SPCE

The effect of pH on Ipa and Epa were also examined in the range of 3.0–10.0. As shown inFigure 4A, when the pH of the solution was increased, Ipa increased gradually and then decayedwith the optimum pH being 7.0. Moreover, considering the fact that physiological pH is around7.0 [26], BR buffer solution of pH 7.0 was used as the supporting electrolyte for the analyses ofthe compounds. Figure 4B illustrates the effect of pH on Epa, which followed the linear regressionequations Epa(V) = 0.51− 0.06 pH, R2 = 0.9998 for UA, Epa(V) = 0.45− 0.04 pH, R2 = 0.9996 forAA, and Epa(V) = 0.72− 0.06 pH, R2 = 0.9995 for DA. The Epa shifted with every increase of thepH value of the supporting electrolyte for all the three compounds, suggesting that the reactions atrGO-SPCE were accompanied by proton transfers [27]. The slope values of 60.0 mV·pH−1 for bothUA and DA suggest that their electrocatalytic reactions involved the transfer of equal number ofprotons and electrons [28]. However, the slope value of 40.0 mV·pH−1 for AA is similar to the valuegiven by the Nernstian equation for reactions involving unequal electron and proton transfers [29];thus, the overall reaction of AA at rGO-SPCE could be classified as an electrochemical process that isfollowed by a chemical reaction [30].

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Figure 4. Effect of pH on the: (A) Ipa; and (B) Epa of UA (0.5 mM), AA (5.0 mM) and DA (0.5 mM) at

rGO-SPCE. (C) Cyclic voltammograms of ternary mixture of UA (0.5 mM), AA (5.0 mM) and DA

(0.5 mM) at SPCE and rGO-SPCE. Supporting electrolyte was BR buffer (pH 7.0), 50 mV·s−1 scan rate.

3.5. CV Analysis of Ternary Mixture of UA, AA and DA at SPCE and rGO-SPCE

The electrochemical behaviour of a ternary mixture of UA, AA and DA at SPCE and rGO-SPCE

was evaluated by CV in order to further understand the electrochemical properties of the electrodes.

At the SPCE, shown in Figure 4C, only two slightly overlapping anodic peaks were obtained at ~0.18

and ~0.287 V; thus, the Epa for UA, AA and DA were indistinguishable, indicating poor selectivity

and sensitivity. In contrast, three well-resolved Epa were observed at 0.183 (for UA), 0.273 (for AA)

and 0.317 V (for DA) on rGO-SPCE. The ∆Ep for UA–AA and AA–DA were 90 mV and 44 mV,

respectively, which is large enough to allow for simultaneous analysis of the compounds in their

ternary mixtures.

3.6. DPV Analysis of Ternary Mixtures of UA, AA and DA at rGO-SPCE

Differential pulse voltammetry (DPV), a widely utilized analytical method for the enhancement

of electrode specificity and sensitivity [30], was carried out by varying the concentration of one of the

compounds while the concentrations of the other two remained constant. As shown in Figure 5A–C,

the Ipa for the three molecules increased linearly with increasing concentrations, suggesting a stable

and efficient electrocatalytic activity at the rGO-SPCE. For UA detection, 2.5 mM AA and 50.0 μM

DA are mixed in BR buffer (pH 7.0) and the corresponding linear regression equation is defined as

𝐼𝑝𝑎 (𝜇𝐴) = 0.03 [𝑈𝐴](𝜇𝑀) + 23.09, 𝑅2 = 0.9998 ([𝑈𝐴] = 10 𝜇𝑀 − 3000 𝜇𝑀). For AA detection,

250.0 μM UA and 50.0 μM DA are mixed in BR buffer (pH 7.0) and the corresponding linear

Figure 4. Effect of pH on the: (A) Ipa; and (B) Epa of UA (0.5 mM), AA (5.0 mM) and DA (0.5 mM)at rGO-SPCE. (C) Cyclic voltammograms of ternary mixture of UA (0.5 mM), AA (5.0 mM) and DA(0.5 mM) at SPCE and rGO-SPCE. Supporting electrolyte was BR buffer (pH 7.0), 50 mV·s−1 scan rate.

3.5. CV Analysis of Ternary Mixture of UA, AA and DA at SPCE and rGO-SPCE

The electrochemical behaviour of a ternary mixture of UA, AA and DA at SPCE and rGO-SPCEwas evaluated by CV in order to further understand the electrochemical properties of the electrodes.At the SPCE, shown in Figure 4C, only two slightly overlapping anodic peaks were obtained at ~0.18and ~0.287 V; thus, the Epa for UA, AA and DA were indistinguishable, indicating poor selectivity andsensitivity. In contrast, three well-resolved Epa were observed at 0.183 (for UA), 0.273 (for AA) and0.317 V (for DA) on rGO-SPCE. The ∆Ep for UA–AA and AA–DA were 90 mV and 44 mV, respectively,which is large enough to allow for simultaneous analysis of the compounds in their ternary mixtures.

3.6. DPV Analysis of Ternary Mixtures of UA, AA and DA at rGO-SPCE

Differential pulse voltammetry (DPV), a widely utilized analytical method for the enhancementof electrode specificity and sensitivity [30], was carried out by varying the concentration of oneof the compounds while the concentrations of the other two remained constant. As shown inFigure 5A–C, the Ipa for the three molecules increased linearly with increasing concentrations,suggesting a stable and efficient electrocatalytic activity at the rGO-SPCE. For UA detection, 2.5 mMAA and 50.0 µM DA are mixed in BR buffer (pH 7.0) and the corresponding linear regressionequation is defined as Ipa (µA) = 0.03 [UA](µM) + 23.09, R2 = 0.9998 ([UA] = 10 µM− 3000 µM).For AA detection, 250.0 µM UA and 50.0 µM DA are mixed in BR buffer (pH 7.0) and the

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corresponding linear regression equation is defined as Ipa (µA) = 12.90 [AA](mM) + 8.55,R2 = 0.9996 ([AA] = 0.1 mM− 2.5 mM), Ipa (µA) = 2.02 [AA](mM) + 56.51, R = 0.9985([AA] = 5.0 mM− 20 mM).

For DA detection, 250.0 µM UA and 2.5 mM AA are mixed in BR buffer (pH 7.0) andthe corresponding linear regression equation is defined as Ipa (µA) = 0.97 [DA](µM) +

2.26, R = 0.9984 ([DA] = 0.20 µM− 80.00 µM), Ipa (µA) = 0.13 [DA](mM) + 75.56, R =

0.9975 ([DA] = 120.0 µM− 500 µM). The calculated limits of detection (S/N = 3) for UA, AA and DAwere found to be 0.4 µM, 50.0 µM and 0.1 µM, respectively. These linear ranges and detection limitsare better than some of the results obtained from the analysis of the compounds using similar modifiedelectrodes (Table 2) and were deemed to be satisfactory for routine analysis of UA, AA and DA.

Chemosensors 2016, 4, 25 8 of 12

regression equation is defined as 𝐼𝑝𝑎 (𝜇𝐴) = 12.90 [𝐴𝐴](𝑚𝑀) + 8.55, 𝑅2 = 0.9996 ([𝐴𝐴] = 0.1 𝑚𝑀 −

2.5 𝑚𝑀), 𝐼𝑝𝑎 (𝜇𝐴) = 2.02 [𝐴𝐴](𝑚𝑀) + 56.51, 𝑅² = 0.9985 ([𝐴𝐴] = 5.0 𝑚𝑀 − 20 𝑚𝑀).

For DA detection, 250.0 μM UA and 2.5 mM AA are mixed in BR buffer (pH 7.0) and the

corresponding linear regression equation is defined as 𝐼𝑝𝑎 (𝜇𝐴) = 0.97 [𝐷𝐴](𝜇𝑀) + 2.26, 𝑅² =

0.9984 ([𝐷𝐴] = 0.20 𝜇𝑀 − 80.00 𝜇𝑀), 𝐼𝑝𝑎 (𝜇𝐴) = 0.13 [𝐷𝐴](𝑚𝑀) + 75.56, 𝑅² = 0.9975 ([𝐷𝐴] =

120.0 𝜇𝑀 − 500 𝜇𝑀). The calculated limits of detection (S/N = 3) for UA, AA and DA were found to

be 0.4 μM, 50.0 μM and 0.1 μM, respectively. These linear ranges and detection limits are better than

some of the results obtained from the analysis of the compounds using similar modified electrodes

(Table 2) and were deemed to be satisfactory for routine analysis of UA, AA and DA.

Figure 5. DPVs of UA, AA and DA at rGO-SPCE in BR buffer (pH 7.0), 50 mV·s−1 scan rate: (A) UA

concentrations of 10.0, 100.0, 250.0, 750.0, 1000.0, 2500.0 and 3000.0 μM in the presence of 2.5 mM AA

and 50.0 μM DA; (B) AA concentrations of 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 5, 7.5, 10.0, 15.0, and 20.0 mM in

the presence of 250.0 μM UA and 50.0 μM DA; and (C) DA concentrations of 0.2, 20.0, 30.0, 40.0, 50.0,

70.0, 80.0, 120.0, 200.0, 250.0, 400.0 and 500.0 μM in the presence of 250.0 μM UA and 2.5 mM AA.

Inserts: Ip vs. concentration.

Figure 5. DPVs of UA, AA and DA at rGO-SPCE in BR buffer (pH 7.0), 50 mV·s−1 scan rate: (A) UAconcentrations of 10.0, 100.0, 250.0, 750.0, 1000.0, 2500.0 and 3000.0 µM in the presence of 2.5 mM AAand 50.0 µM DA; (B) AA concentrations of 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 5, 7.5, 10.0, 15.0, and 20.0 mM in thepresence of 250.0 µM UA and 50.0 µM DA; and (C) DA concentrations of 0.2, 20.0, 30.0, 40.0, 50.0, 70.0,80.0, 120.0, 200.0, 250.0, 400.0 and 500.0 µM in the presence of 250.0 µM UA and 2.5 mM AA. Inserts:Ip vs. concentration.

Chemosensors 2016, 4, 25 9 of 12

Table 2. Comparison of the analytical performance characteristics of UA, AA and DA electrodes.

Electrode Material Limit of Detection (µM) Linear Range (µM) Reference

UA AA DA UA AA DA

ERGO-GCE 0.5 300.0 0.5 0.5–60.0 500.0–2000.0 0.5–60.0 [31]

GCE 4.7 23.38 2.67 5.0–70.0 25,000.0–300,000.0 3.0–30.0 [32]

Trp-GR 1.24 10.09 0.29 10.0–1000.0200.0–3400.0,

0.5–110.0 [33]3400.0–12,900.0

PdNPs-GR-CS-GCE 0.17 20.0 0.1 0.5.0–200.0 100.0–400.00.50–15.0,

[34]20.00–200.00

AgNPs-rGO-GCE 8.2 9.6 5.4 10.0–800.0 10.0–800.0 10.00–800.00 [35]

MWCNT-GCE 0.42 7.71 0.31 0.55–90.0 15.0–800.0 0.50–100.00 [36]

Nitrogen doped GR 0.045 2.2 0.25 0.1–20.0 5.0–1300.0 0.50–170.00 [29]

poly(Tyr)-MWCNTs-COOH-GCE 0.3 2.0 0.02 1.0–350.0 50–1000.0 0.10–30.00 [37]

Screen printed graphene electrode 0.2 0.95 0.12 0.8–2500.0 4.0–4500.0 0.50–2000.00 [38]

HCNTs-GCE 1.5 0.92 0.8 6.7–65.0 7.5–180.0 2.50–105.00 [39]

IL-GR-GCE 0.323 – 0.679 1.0–600.0 – 1.00–400.00 [40]

rGO-SPCE 0.35 50 0.1 10.0–3000.0100.0–2500.0, 0.20–80.00, This study

5000.0–20000.0 120.0–500.0

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3.7. Reproducibility and Stability of rGO-SPCE, and Ternary Mixture Analysis

In order to evaluate the reproducibility of rGO-SPCE, a single rGO-SPCE was used (n = 6) todetermine the concentrations of the compounds in a ternary mixture (250.0 µM UA, 2.5 mM AA,and 50.0 µM DA) by DPV. It was found that the relative standard deviations for the peak currentintensities for UA, AA and DA were 1.05%, 1.51% and 1.25%, respectively. The stability of rGO-SPCEwas also determined in a similar fashion after storage in room temperature for 21 days; the peakcurrent intensities for UA, AA and DA decayed by less than 2.5%, suggesting that the electrodeswere highly stable over the 21-day period. The rGO-SPCE was also used to analyse ternary mixturesof the compounds in urine using standard addition method. The analytical performance data aresummarized in Table 3. The recoveries were in the range of 98.07% to 99.97% with a relative standarddeviation (RSD, n = 3) of less than 1.5%. Clearly, the presence of common interferences in the urinesamples did not interfere with the analysis of the compounds; thus, the sensor can be used for routinesimultaneous quantification of the compounds in biological samples.

Table 3. Recovery of UA, AA and DA from ternary mixtures in urine.

Sample Amount Added (µM) Amount Recovered (µM) Mean Recovery (%)

UA - - -

Repeat 1 50 47.9Repeat 2 50 49.4Repeat 3 50 49.8

Mean - 49.033 98.07

AA - - -

Repeat 1 500 497.3Repeat 2 500 499.7Repeat 3 500 499.5

Mean - 499.833 99.97

DA - -

Repeat 1 50 49.4Repeat 2 50 48.8Repeat 3 50 49.9

Mean - 49.367 98.73

4. Conclusions

An in-house fabricated screen-printed carbon electrode, modified with chemically reducedgraphene oxide, has been demonstrated as a useful analytical sensing tool for simultaneous analysis ofuric acid, ascorbic acid and dopamine. The sensor showed stable responses for repeated measurementsand good electrocatalytic capability that enabled the resolution of three voltammetric peaks associatedwith the compounds, thus enabling their simultaneous analyses. The sensor could be employed forroutine simultaneous analysis of UA, AA and DA.

Acknowledgments: The authors would like to thank the Department of Employment and Learning Ireland (GrantNo.: USI035) and the National Institutes of Health (Grant No.: 5R01ES003154-30) for the funding.

Author Contributions: Prosper Kanyong conceived, designed and performed the experiments, analyzed thedata, and wrote the paper. Sean Rawlinson designed and printed the electrodes. James Davis provided reagents,materials and equipment, and supervised the project.

Conflicts of Interest: The authors declare no conflict of interest.

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